Sequences from Piscirickettsia salmonis

ABSTRACT

The present invention relates to a fish vaccine. More specifically the invention relates to a vaccine to protect salmon against infection by  Piscirickettsia salmonis . The invention is based on or derived from the nucleic acid or amino acid sequence of antigens from  Piscirickettsia salmonis . Nucleic acid and/or amino acid sequences may be used in the preparation of a vaccine to protect against infection by  Piscirickettsia salmonis.

This application is a continuation-in-part of International PCTApplication No. PCTIGB 01/01055, file Mar. 12, 2001, which in itsentirety is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a fish vaccine. More specifically theinvention relates to a vaccine to protect salmon againstpiscirickettsiosis, also referred to as salmonid rickettsial septicaemia(SRS).

BACKGROUND OF THE INVENTION

To date no commercially available vaccine has succeeded in controllinginfections by Piscirickettsia salmonis, the causative agent of SRS.Accordingly there is a need for an effective vaccine againstPiscirickettsia salmonis.

It is an object of the present invention to provide a protein basedvaccine or a Nucleic Acid Vaccine (NAV) to protect fish againstinfection by Piscirickettsia salmonis, and thereby against SRS.

SUMMARY OF THE INVENTION

In one aspect of the invention, novel nucleic acid sequences fromPiscirickettsia salmonis and their encoded amino acid sequences areprovided.

In another aspect of the invention a method is provided for protectingfish against infection by Piscirickettsia salmonis, comprisingadministering to a fish one or more of the nucleic acid sequences andamino acid sequences of the invention.

A further aspect of the invention provides nucleic acid vaccines andprotein vaccines comprising one or more of the nucleic acid sequences oramino acid sequences of the invention, for administration to fish toprotect against SRS.

DETAILED DESCRIPTION OF THE INVENTION

The novel sequences of the genes of the invention and the encodedproteins are provided in SEQ ID NOS: 1 through 34 which are containingthe Sequence Listing.

Psclone51A

A partial nucleotide seqyence of Psicone51A is contained in SEQ ID NO:1and complete nucleotide sequence of Psclone51A are contained in SEQ IDNO:3. These sequences were obtained from a cDNA molecule from mRNAofPiscirickettsia salmonis type strain LF-89. (cat. no. VR-1361, ATCC,Manassas, Va.). The cloned material was sequenced in both directionsfrom the 5′ and 3′ insertion sites using overlapping amplicons. PCR andRT-PCR comparing uninfected Chinook salmon embryonic (CHSE-214) celllines and cell lines infected with P. salmonis confirmed that Psclone51Ais specific to P. salmonis and is not derived from salmon host cells.

The amino acid sequence of the open reading frame (ORF) of the partialnucleotide sequence of Psclone51A is contained in SEQ ID NO:2. The aminoacid sequence of the ORF of the complete nucleotide sequence ofPsclone51A is contained in SEQ ID NO:4. Expression of the clonedsequence has yielded a protein of approximately 12 kDa.

The ORF of Psclone51A in SEQ ID NO: 3 does not have any significanthomology at the nucleotide level with previous submissions to databasesaccessible by BLAST. At the protein level, a border line similarity witha hypothetical 21.5 kDa protein of Escherichia coli was found.

p10.6

An expression library was constructed from Piscirickettsia salmonisstrain LF-89 cat. no. VR-1361, ATCC, Manasses, Va.), grown in CHSE-214cells. An immuno-reactive clone, (0110-2-5) (p10.6), was selected usinghomologous antisera from Piscirickettsia salmonis immunised rabbits andsequenced. The nucleotide sequence of 10.6 is in SEQ ID NO:5. The openreading frame (ORF) for the full gene was completed by inverse PCR fromgenomic Piscirickettsia salmonis DNA.

Polymerase chain reaction (PCR) primers were designed based on thesequence of clone p10.6. Using these primers, PCR and RT-PCR comparinguninfected Chinook salmon embryonic (CHSE-214) cell lines and cell linesinfected with P. salmonis confirmed that clone p10.6 is specific to P.salmonis and is not derived from salmon host cells.

The amino acid sequence deduced for the open reading frame (ORF) is inSEQ ID NO:6. The ORF of p10.6 does not have any significant homology atthe nucleotide level with previous submissions to databases accessibleby BLAST. The derived amino acid sequence, but not the nucleotidesequence, shows significant homology to the 17 kDa antigen found inRickettsia of the Spotted Fever Group, where it is considered a groupspecific, outer membrane protein.

IcmE

The nucleic acid sequence of IcmE is in SEQ ID NO:7). The geneticsequence has been derived from an inverse polymerase chain reaction(IPCR) product amplified from Piscirickettsia salmonis type strain LF-89genomic DNA(gDNA). The IPCR product was sequenced in both direction fromthe 5′ and 3′ sides using overlapping amplicons.

The amino acid sequence deduced for the ORF is provided in SEQ ID NO:8.The protein encoded by the ORF of IcmE (403) has a 37% significanthomology at the protein level to the IcmE protein of Legionellapneumophila when compared to previous submissions to databasesaccessible by BLAST.

p45

The amino acid sequence of a portion of the p45 major antigen of P.salmonis is provided in SEQ ID NO:9.

The amino acid sequence was derived from microsequencing of a proteinapproximately 45 kDa found to be immunoreactive to rabbit anti-Psalmonis antibodies. Moreover, p45 was found uniquely in Chinook salmonembryonic (CHSE-214) cells infected with Piscirickettsia salmonis andnot in uninfected CHSE-214 cells.

The amino acid sequence of p45 has no significant homology to otherbacterial proteins when compared to previous submissions to databasesaccessible by BLAST.

The fragment of p45 provided in SEQ ID NO:9 can be used per se inpreparation of an antigen-based vaccine. Alternatively, a nucleic acidsequence encoding this fragment can be employed in the context of a DNAvaccine against P. salmonis. The p45 sequence information can also beused to isolate and sequence genomic or cDNA clones from P. salmonis inorder to enable production of a vaccine based on the complete nucleicacid sequence or amino acid sequence of p45.

clone 3

The following three plasmids have related nucleic acid sequences:clone3/original, clone3/3PST-R, and clone3/3APA-F. For clone3/originalthe nucleotide sequence is in SEQ ID NO:10 and the amino acid sequenceencoded therein is in SEQ ID NO:11. For clone3/3PST-R the nucleotidesequence is found in SEQ ID NO:12 while the amino acid sequence encodedtherein is in SEQ ID NO:13. For clone3/3APA-F the nucleotide sequence isin SEQ ID NO:14 while the amino acid sequence encoded therein is foundin SEQ ID NO:15.

The proteins encoded by the ORF of clone3/original, clone3/3PST-R andclone3/3APA-F have respectively 40%, 38% and 34% significant homology atthe protein level to different portion of the transposase protein ofVibrio anguillarum (NCBI Protein Database number AAA81776.1) whencompared to previous submissions to databases accessible by BLAST.

clone 7

The following four plasmids have related nucleic acid sequences:clone7/original, clone7/XbaR, clone7/MunR, and clone 7/MunF. Forclone7/original, the nucleotide sequence is contained in SEQ ID NO:16and the amino acid sequence encoded thereby is in SEQ ID NO:17. Forclone7/XbaR, the nucleotide sequence is contained in SEQ ID NO:18 and inamino acid sequence encoded thereby is in SEQ ID NO:19. For theclone7/MunR, the nucleotide sequence is contained in SEQ ID NO:20 andthe amino acid sequence encoded thereby is in SEQ ID NO:21. Forclone7/MunF, the nucleotide sequence is contained in SEQ ID NO:22 andthe amino acid sequence encoded thereby is in SEQ ID NO:23.

The genetic sequences have been derived from an inverse polymerase chainreaction (IPCR) product amplified from Piscirickettsia salmonis genomicDNA (gDNA).

The peptides encoded by the ORF of clone7/original, clone7/XbaR,clone7/MunR, and clone 7/MunF have a 40% to 44% significant homology atthe protein level to different portion of the ABC transporterATP-binding protein of the other bacterial species when compared toprevious submissions to databases accessible by BLAST.

There is sufficient reason to believe that the nucleotide andcorresponding amino acid sequence are of Piscirickettsia salmonisorigin. Also, part of the ORFs was found in an immuno-reactive clone ofan expression library.

clone 20

Related nucleic acid sequences are clone20/original (SEQ ID NO:24), andclone20/20VSPF (SEQ ID NO: 26). The encoded amino acid sequences are SEQID NO:25 and SEQ ID NO: 27 respectively.

The genetic sequences have been derived from an inverse polymerase chainreaction (IPCR) product amplified from Piscirickettsia salmonis genomicDNA (gDNA).

The peptides encoded by the ORF of clone20/original, and clone20/20VSPFhave a 41% and 51% significant homology at the protein level to an aminoacid transporter/permase protein of other organisms when compared toprevious submissions to databases accessible by BLAST.

clone 15

The nucleic acid sequence of clone15/original is contained in SEQ ID NO:28 and the encoded amino acid sequence thereof is contained in SEQ IDNO: 29.

The nucleotide sequence and the peptide encoded by the ORF ofclone15/original have no significant homology to proteins of otherbacterial species when compared to previous submissions to databasesaccessible by BLAST.

From the previous information there is sufficient reason to believe thatthe nucleotide and corresponding amino acid sequence are ofPiscirickettsia salmonis origin. Also, the ORF was found in animmuno-reactive clone of an expression library.

Hsp70

The amino acid sequence of Hsp70 is contained in SEQ ID NO: 30. Theamino acid sequence obtained from another Hsp70 isolate (clone B) iscontained in SEQ ID NO:31, and the nucleic acid sequence encoding thatamino acid sequence is in SEQ ID NO:32.

The amino acid sequence of hsp70 has 70% significant homology at theprotein level to Coxiella burnetti and Legionella pneumophila hsp70proteins when compared to previous submissions to databases accessibleby BLAST.

Hsp60

SEQ ID NO:33 contains the nucleic acid sequence of the hsp60 gene of P.salmonis. The amino acid sequence deduced from the nucleic acid sequenceis contained in SEQ ID NO:34.

Characterization of the P. salmonis hsp60 gene was initiated byamplifying a 600 bp product from P. salmonis mRNA using universaldegenerate primers. The primers are as described in Swee Han Goh etal.(1996) Journal Clin. Microbiol. 34(4): 818-823. The PCR product wassequenced by standard methods (Molecular Cloning: A Laboratory Manual(1989)2^(nd) ed. Sambrook, J. et al., Cold Spring Harbor Laboratory,NY).

Once this first PCR product was sequenced, the sequence of the ORF wascompleted by performing several inverse PCR (IPCR) amplifications ongenomic DNA from Piscirickettsia salmonis type strain LF-89 (Triglia etal, 1988, Nucl. Acids Res. 16: 8186). The IPCR products were sequencedin both directions from the 5′ and 3′ insertion sites using overlappingamplicons.

The nucleic acid sequence and protein sequence of P. salmonis hsp60 showsimilarity to the corresponding gene/protein in other species. Theprotein encoded by the ORF of P. salmonis hsp60 has a 75-77% significanthomology to the GroEL protein of various Pseudomonas and Vibrio specieswhen compared to previous submissions to databases accessible by BLAST.

Heat shock proteins are abundant molecules which participate in folding,assembly and disassembly of protein complexes. Hsp60 and Hsp70 are majortargets of immune responses to a wide variety of pathogens, includingbacteria, fungi, helminths and protozoan parasites. It has beendemonstrated that immunization with certain pathogen hsps induces strongimmune responses and provides protection against disease caused by thesepathogens. The strength of these immune responses may reflect theabundance of these proteins, and their immunogenicity: multiple B celland T cell epitopes are found on mycobacterial hsp60.

Consequently, there are grounds for believing that P. salmonis hsp70 andhsp60 can form the basis for a successful monovalent or multivalentvaccine targeted against SRS and related diseases.

The gene and protein sequences of the invention are preferably derivedfrom P. salmonis type strain LF89. The invention encompasses nucleicacid sequences and amino acid sequences which are substantiallyhomologous to the sequences provided in the Figures. “Substantiallyhomologous” in this context means that a sequence, when compared to areference sequence, has at least 60% homology, preferably at least 70%homology, more preferably at least 80% homology, more preferably atleast 90% homology, and most preferably at least 95% homology to thereference sequence.

To determine the percent homology of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g. gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence and the intervening non-homologoussequence in the gap can be disregarded for comparison purposes). Thereis no requirement for the two sequences to be the same length. Ingeneral, the length of sequence across which the sequences are comparedis the entire extent of the alignment. Optionally, the length of areference sequence aligned for comparison purpose is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least, 70%, 80%, or90% of the length of the reference sequence. It possible to restricthomology analysis to any particular portion of the reference sequence.

When a position in the first (reference) sequence is occupied by thesame amino acid residue or nucleotide as the corresponding position inthe sequence, the molecules are homologous at that position (i.e. thereis identity at that position). In the case of nucleic acid sequencecomparison there is also homology at a certain position where the codontriplet including the nucleotide encodes the same amino acid in bothmolecules being compared, due to degeneracy of the genetic code.

The percent homology between two sequences is a function of the numberof homologous positions shared by the sequences (i.e., % homology=no. ofhomologous positions/total no. of positions). Optionally, the comparisonof sequences and determination of percent homology can be accomplishedusing a mathematical algorithm. Suitable algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:430-10.

The definition of homologous sequences provided above embraces fragmentsof the reference nucleic acid sequence or amino acid sequence. Forpresent purposes a “fragment” of a protein of the invention isunderstood to mean any peptide molecule having at least 20, optionallyat least 30, or at least 40 contiguous amino acids of the referenceamino acid sequence. A “fragment” of a nucleic acid reference sequenceis any part of that sequence comprising at least 50, optionally at least75, or at least 100 consecutive nucleotides.

Also comprised within the nucleic acid sequences of the invention aresequences which hybridize to the reference nucleic acid sequences understringent conditions. “Stringent” hybridization conditions in the senseof the present invention are defined as those described by Sambrook etal., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press (1989), 1.101-1.104, i.e. a positive hybridizationsignal is still observed after washing for 1 hour with lx SSC buffer and0.1% SDS at 55° C., preferably at 62° C. and most preferably at 68° C.,in particular for 1 hour in 0.2×SSC buffer and 0.1% SDS at 55° C.,preferably at 62° C. and most preferably at 68° C.

In particular, the invention extends to oligonucleotides 15 to 100,preferably 20 to 50, most preferably 25 to 35 nucleotides in length,which are complementary to any of the nucleic acid sequences depicted inthe figures or which are capable of hybridising to these sequences understringent conditions.

The amino acid sequences of the invention also comprise syntheticanalogues or derivatives of the sequences in the Figures, or ofhomologues of those sequences. A “derivative” of an amino acid sequenceis a sequence related to the reference sequence either on the amino acidsequence level (e.g. a homologous sequence wherein certainnaturally-occurring amino acids are replaced with synthetic amino acidsubstitutes) or at the 3D level, i.e. molecules having approximately thesame shape and conformation as the reference amino acid sequence. Thus,derivatives include mutants, mimetics, mimotopes, analogues, monomericforms and functional equivalents. Amino acid sequence derivatives retainthe ability to induce the production of antibodies that recognize and(cross)-react with antigens of P. salmonis and/or to induce an immuneresponse in fish that protects against infection with this pathogen.

The present invention provides the use of any of the nucleic acidsequences or amino acid sequences shown in the Figures, or relatedsequences, in the manufacture of a vaccine for the protection of fishagainst infection by Piscirickettsia salmonis.

The invention further provides a vaccine to protect fish againstPiscirickettsia salmonis wherein the vaccine includes at least onenucleic acid or peptide sequence as defined herein, together with apharmaceutically acceptable carrier.

A diagnostic test kit is also provided in accordance with the invention,whereby the kit may comprise a nucleic acid sequence or amino acidsequence of the invention, or may comprise an antibody capable ofrecognising any of the amino acid sequences of the invention.

The isolated nucleic acid sequences from P. salmonis can be exploited inthe conventional manner, by cloning the gene into an expression vectorfor generation of large quantities of purified or isolated recombinantprotein. A purified antigen can also be obtained by non-recombinanttechniques, i.e. through extraction from cells by conventionalpurification methods. Alternatively, the protein or polypeptide can besynthesized chemically using standard peptide synthesis techniques. Avaccine comprising this purified or isolated recombinant ornon-recombinant protein can be termed an antigen-based vaccine.

An “isolated” or “purified” protein is defined as being substantiallyfree of cellular material or other contaminating proteins from the cellor tissue source from which the purified protein is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of a candidate protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations ofcandidate protein having less than about 30% (by dry weight) ofnon-candidate protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of contaminating protein,still more preferably less than about 10% of contaminating protein, andmost preferably less than about 5% contaminating protein. When thecandidate protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

Alternatively, the genes of the invention can be incorporated intoNucleic Acid Vaccines (NAVs), whereby the NAV is taken up by host cellsof a living animal, and expression of the gene takes place within thecytosol.

A gene inserted into a DNA vector can be inoculated directly into a fish(e.g. orally, intramuscularly or intraperitoneally) for expression invivo within fish cells. Thus, in one aspect of the invention there isprovided a nucleic acid vaccine comprising a pharmaceutically acceptablecarrier and a DNA plasmid in which a nucleic acid sequence encoding a

P. salmonis gene of the invention is operably linked to atranscriptional regulatory sequence. Transcriptional regulatorysequences include promoters, polyadenylation sequences and othernucleotide sequences such as the immune-stimulating oligonucleotideshaving unmethylated CpG dinucleotides, or nucleotide sequences that codefor other antigenic proteins or adjuvanting cytokines. For optimal invivo expression it may be preferred to select transcriptional regulatorysequences endogenous to the fish to be vaccinated. For instance,endogenous cytokine or actin gene promoters may be considered. The DNAcan be present in naked form or it can be administered together with anagent facilitating cellular uptake (e.g. liposomes or cationic lipids).The technology of DNA vaccination of fish is explained in more detail inU.S. Pat. No. 5,780,448, which is incorporated herein by reference.

Another aspect of the invention pertains to vectors, preferablyexpression vectors, comprising a nucleic acid sequencing encoding a geneof the invention (or a portion thereof). As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operatively linked to the nucleicacid sequence to be expressed. Recombinant expression vectors of theinvention may be used for expression within the intended recipient ofthe antigen of the invention (as a DNA vaccine) or for expression withina host organism other than the final recipient (for production ofrecombinant antigen vaccines).

Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, hsp proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Other suitable host cells are known to those skilled inthe art (e.g. Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990)). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Often, infusion expression vectors, a proteolytic cleavage site is introduced atthe junction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

The present invention also relates to a method of generating monoclonalor polyclonal antibodies to an amino acid sequence of the invention. Inthis embodiment, an effective amount of the amino acid sequence (i.e.,an amount which results in an immune response in the host) is introducedinto an animal host which results in production of antibodies to thesubstance in the host. The antibodies are removed from the host andpurified using known techniques (e.g. chromatography), thereby resultingin production of polyclonal antibodies. Procedures for immunizinganimals, eg. mice, with proteins and selection of hybridomas producingimmunogen-specific monoclonal antibodies are well known in the art (seefor example Kohler and Milstein (1975) Nature 256: 495-497). Theantibodies of the invention recognize (have an affinity to) at least oneof the amino acid sequences disclosed herein. Preferably, the antibodiesof the invention are raised against an isolated or purified amino acidsequence of the invention.

The vaccines manufactured in accordance with the methodology of theinvention are suited for administering to any aquatic animal species forpreventative or therapeutic purposes. The vaccines of the invention canbe employed in treatment of teleosts such as salmon (Chinook, Atlantic,Coho), trout (including rainbow trout), carp, sea bream, sea bass,yellowtail, tilapia, grouper, catfish, halibut, haddock, or optionallyfor treatment of other aquatic species such as crustaceans and mollusks.Salmonid fish are the preferred species for treatment.

It is possible to immunize a subject with the neutral or the salt formsof the present purified or isolated proteins, either administered aloneor in admixture with a pharmaceutically acceptable vehicle or excipient.Typically, vaccines are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to administration may also be prepared. The preparation may beemulsified or the active ingredient encapsulated in liposome vehicles.The pharmaceutical compositions of the invention may be administered ina form for immediate release or by extended release.

Pharmaceutically acceptable carriers or vehicles include conventionalexcipients, and may be, for example, solvents such as water, oil orsaline, dextrose, glycerol, wetting or emulsifying agents, bulkingagents, coatings, binders, fillers, disintegrants, diluents, lubricants,pH buffering agents, or conventional adjuvants such as muramyldipeptides, avridine, aluminium hydroxide, oils, saponins, blockcopolymers and other substances known in the art.

To immunize a subject, an antigen or gene vector can be administeredparenterally, usually by intramuscular injection in an appropriatevehicle, but optionally by the subcutaneous route, by intravenousinjection or by intradermal or intranasal delivery. In the case ofimmunization of fish, the typical routes of administration are byinjection into the peritoneal cavity, orally in feed, or by immersion insea water or fresh water. Consequently it is preferred to administer theamino acid sequences and nucleic acid sequences of the invention in theform of oral or injectable formulations, or as a liquid (for instance aliquid emulsion or emulsifiable concentrate) to be added to a water tankor bath where the fish are held.

The effective dosage may vary depending on the size and species of thesubject, and according to the mode of administration. The optimal dosagecan be determined through trial and error by an aquaculture specialistor veterinarian. Typically, a single dose of antigen will be in therange of from about 0.01 to 1000 μg per kg body weight, preferably 0.5to 500 μg per kg, more preferably 0.1 to 100 μg per kg. For DNAvaccines, a minimum dosage of 10 pg up to dosages of 1000 pg of plasmidper animal should be sufficient for suitable expression of the antigenin vivo.

The novel antigens disclosed as part of the present invention are alsouseful in screening for antibodies to pathogenic proteins. The inventionadditionally includes diagnostic uses of these antigens, for instance inthe preparation of a diagnostic kit, useful for testing animals for thepresence of disease-causing organisms.

It is also contemplated that antibodies raised against the purifiedantigens of the invention can have both diagnostic and therapeuticapplications in disease management. Both polyclonal antibodies andmonoclonal antibodies may be useful in this respect. Sandwich assays andELISA may be mentioned as specific examples of diagnostic assays.

EXAMPLES Example 1

Efficacy of nucleic acid vaccines comprising P. salmonis antigensequences

Coho salmon parr (Oncorhynchus kisutch) less than 6 months old and ofaverage weight 5.4 grams were obtained from a disease free stock andwere acclimatised to water at a temperature of 9±1° C., flowing at arate of 2.5 L/min. Stocking densities were maintained at <20 kg/m³, andthe fish were fed a commercial pelleted diet at a daily rate of 1.5%body weight. The weight of fish in all groups was recorded prior to eachvaccination and at the end of the trial. Any behavioural changes in thefish were also noted on a daily basis.

Coho salmon are particularly susceptible to infection with P. salmonis.

TABLE 1 sets out the experimental design: Group Size Treatment DoseRoute 1 110 pUK blank 25 μg i.m. 2 110 pUK-Psclone51A 25 μg i.m. 3 110pUK-Pshsp60 25 μg i.m. 4 110 pUK-Ps17kD 25 μg i.m. 5 110 P. salmonis/oil0.1 ml i.p. 6 110 PBS 0.1 ml i.m.

The treatments were administered to randomly allocated groups of fishanaesthetized with 50 mg/L benzocaine, by single injection via theintramuscular (i.m.) route or intraperitoneal (i.p.) route.

pUK is an expression vector backbone which was not expected to induceany protection against SRS. pUK-psclone51A is a NAV construct carryingthe entire ORF of Psclone51A. pUK-PsHSP60 is a NAV construct carryingthe entire ORF of the P. salmonis hsp60 gene. pUK-Ps17kD is a NAVconstruct carrying the entire ORF of the P. salmonis p10.6 gene. Thepositive control group 4 was injected with a preparation of inactivatedP. salmonis (strain LF89). This preparation is known to elicitprotection against SRS, but is too expensive to produce on a commercialscale.

Following immunisation the fish were returned to holding tanks and keptthere for 600 degree days before challenge. At this time fish from eachtreatment group were randomly divided into challenge tanks and controltanks. Treated fish were challenged by intraperitoneal injection with0.1 ml containing approximately 10^(3.5) TCID₅₀ of cultured P. salmonis.Fish in each tank were monitored daily for mortality. Each mortalityevent was investigated for evidence of P. salmonis infection (by PCR).

Example 2

Immunogenicity of Antigens

Hsp60, Hsp70 and p10.6 nucleic acid sequences were inserted into aconventional expression vector. The recombinant proteins expressed in E.coli were purified and injected into mice in conjunction with anadjuvant. 40 days later, the mice were sacrificed and tested by ELISAfor production of antibodies specific to the injected antigens. In everycase, a specific immune response had been mounted to the antigen. Asimilar result was obtained when a plasmid NAV construct carrying thehsp70 gene was injected into mice.

These data provide solid evidence that the recombinant proteins Hsp60,Hsp70 and p10.6 are highly immunogenic, and are likely to be capable ofinducing an immune response in fish, specifically targeting P. salmonisand thereby preventing development of SRS.

1. An isolated nucleic acid sequence comprising SEQ ID NO:
 33. 2. Theisolated nuclei acid sequence of claim 1 whereby said isolated nucleicacid sequence is isolated from Piscirickettsia salmonis type strainLF-89.
 3. An isolated nucleic acid sequence having at least 80% homologythe sequence set forth in to SEQ ID NO: 33, whereby said isolated nucleiacid sequence can detect Piscirickettsia salmonis DNA by stringenthybridization conditions, wherein said stringent conditions comprise 1XSSC, 0.1% SDS at 55° C.
 4. A DNA expression vector comprising a nucleicacid of claim
 1. 5. An isolated nucleic acid sequence encoding the aminoacid sequence set forth in SEQ ID NO:
 34. 6. An isolated polynucleotidefully complementary to the nucleic acid sequence of claim
 1. 7. Adiagnostic test kit for detecting Piscirickettsia salmonis DNScomprising an isolated polynucleotide of claim
 6. 8. An isolatedpolynucleotide fully complementary to the nucleic acid sequence of claim3.